Role of the RNA modifications 2’-O-methylation and N6-methyladenosine in viral pathogenicity

Research area: Molecular Immunology

Viral RNA is sensed by various pattern recognition receptors such as Toll-like receptors and RIG-I like helicases (RLH) leading to immune activation and type I interferon production. Several viral evasion strategies targeting signal transduction or modifying 5’ RNA ends have been described. However, RNA modifications such as 2’-O-methylation or N6-methyladenosine that antagonize or negatively modulate TLR7-mediated RNA recognition have not been considered. This proposal addresses the possible role of host cell-derived 2’-O-methylations in genomic influenza A virus (IAV) RNA and predicted host-specific methylation differences for altered immune response, infectivity and pathogenicity. In addition, we will analyze the effect of antagonistic 2’-O-methylated host cell tRNA, that is associated with VSV or SeV, on early immune escape from TLR7 recognition in plasmacytoid dendritic cells. Importantly, we will also investigate the currently unknown function of N6-methyladenosine  (m6A) in influenza mRNA. Since the number and position of predicted m6A methylation sites in IAV mRNA strongly vary among different IAV strains, we hypothesize an influence on immune recognition, viral replication and/or pathogenicity.

Overall, this analysis will provide a more sophisticated understanding of the immune response to virus infection or pathogenicity and may lead to new approaches for antiviral drug development.

Project-related publications of the investigator:

  • Jung S, von Thülen T, Laukemper V, Pigisch S, Hangel D, Wagner H, Kaufmann A, Bauer S. A single naturally occurring 2′-O-methylation converts a TLR7- and TLR8-activating RNA into a TLR8-specific ligand. PLoS One. 2015 Mar 18;10(3):e0120498. doi:10.1371/journal.pone.0120498. eCollection 2015.
  • Oldenburg M, Krüger A, Ferstl R, Kaufmann A, Nees G, Sigmund A, Bathke B, Lauterbach H, Suter M, Dreher S, Koedel U, Akira S, Kawai T, Buer J, Wagner H, Bauer S, Hochrein H, Kirschning CJ. TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification. Science. 2012 Aug 31;337(6098):1111-5. doi: 10.1126/science.1220363. Epub 2012 Jul 19.
  • Bauer, S., C. J. Kirschning, H. Hacker, V. Redecke, S. Hausmann, S. Akira, H. Wagner, and G. B. Lipford. 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci U S A 98:9237-42.
  • Hamm, S., A. Heit, M. Koffler, K. M. Huster, S. Akira, D. H. Busch, H. Wagner, and S. Bauer. 2007. Immunostimulatory RNA is a potent inducer of antigen-specific cytotoxic and humoral immune response in vivo. Int Immunol 19:297-304.
  • Hamm, S., E. Latz, D. Hangel, T. Muller, P. Yu, D. Golenbock, T. Sparwasser, H. Wagner, and S. Bauer. 2010. Alternating 2′-O-ribose methylation is a universal approach for generating non-stimulatory siRNA by acting as TLR7 antagonist. Immunobiology 215:559-69.
  • Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, G. Lipford, H. Wagner, and S. Bauer. 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526-9.
  • Heil, F., P. Ahmad-Nejad, H. Hemmi, H. Hochrein, F. Ampenberger, T. Gellert, H. Dietrich, G. Lipford, K. Takeda, S. Akira, H. Wagner, and S. Bauer. 2003. The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol 33:2987-97.
  • Jöckel, S., G. Nees, R. Sommer, Y. Zhao, D. Cherkasov, H. Hori, G. Ehm, M. Schnare, M. Nain, A. Kaufmann, and S. Bauer. 2012. The 2′-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition. J Exp Med 209:235-41.
  • Jurk, M., F. Heil, J. Vollmer, C. Schetter, A. M. Krieg, H. Wagner, G. Lipford, and S. Bauer. 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 3:499.
  • Rutz, M., J. Metzger, T. Gellert, P. Luppa, G. B. Lipford, H. Wagner, and S. Bauer. 2004. Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol 34:2541-50.

Control of Hepatitis C Virus replication by viral and cellular factors

Research area: Molecular Virology

Replication of Hepatitis C Virus (HCV) genome RNA in cells requires both the establishment and specific activity of viral RNA replication complexes and also viral countermeasures against the cellular innate immune system. We found that HCV stimulates expression of a long non-coding RNA (Lnc-ITM2C-1 or GCSIR) that in turn stimulates expression of a cannabinoid receptor, GPR55. This in turn suppresses expression of interferon-stimulated genes (ISGs) in favour of HCV replication. In this project, we aim at gaining deeper insight into the molecular mechanisms of the Lnc-ITM2C-1 – GPR55 – ISG regulation axis. Complementary to the early steps of the cellular response, we aim at elucidating the early steps of HCV RNA synthesis in cells, using full-length and subgenomic replicon systems. Ribosome pausing at two positions at the NS5B replicase stop codon and directly upstream is caused by inefficient codons, indicating that a slow-down of NS5B translation may be important for the initiation of minus strand synthesis. We want to identify HCV RNA cis-signals and long-range interactions involved in the balance of initial RNA genome translation starting at the genome´s 5´-end versus the initiation of RNA minus strand synthesis starting at the genome´s 3´-end.

Project-related publications of the investigator:

  • Niepmann M, Gerresheim GK. 2020. Hepatitis C Virus Translation Regulation. Int J Mol Sci 21.
  • Gerresheim GK, Hess CS, Shalamova LA, Fricke M, Marz M, Andreev DE, Shatsky IN, Niepmann M. 2020. Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication. Int J Mol Sci 21.
  • Hu P, Wilhelm J, Gerresheim GK, Shalamova LA, Niepmann M. 2019. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses 11.
  • Gerresheim GK, Roeb E, Michel AM, Niepmann M. 2019. Hepatitis C Virus Downregulates Core Subunits of Oxidative Phosphorylation, Reminiscent of the Warburg Effect in Cancer Cells. Cells 8.
  • Gerresheim GK, Bathke J, Michel AM, Andreev DE, Shalamova LA, Rossbach O, Hu P, Glebe D, Fricke M, Marz M, Goesmann A, Kiniry SJ, Baranov PV, Shatsky IN, Niepmann M. 2019. Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling. Int J Mol Sci 20.
  • Fricke M, Gerst R, Ibrahim B, Niepmann M, Marz M. 2019. Global importance of RNA secondary structures in protein-coding sequences. Bioinformatics 35:579-583.
  • Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O. 2018. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol 9:395.
  • Jost I, Shalamova LA, Gerresheim GK, Niepmann M, Bindereif A, Rossbach O. 2018. Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges. RNA Biol 15:1032-1039.
  • Nieder-Rohrmann A, Dunnes N, Gerresheim GK, Shalamova LA, Herchenrother A, Niepmann M. 2017. Cooperative enhancement of translation by two adjacent microRNA-122/Argonaute 2 complexes binding to the 5′ untranslated region of hepatitis C virus RNA. J Gen Virol 98:212-224.
  • Gerresheim GK, Dunnes N, Nieder-Rohrmann A, Shalamova LA, Fricke M, Hofacker I, Honer Zu Siederdissen C, Marz M, Niepmann M. 2017. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure. Cell Mol Life Sci 74:747-760.

Replication of large RNA virus genomes: key enzymes and mechanisms

Research area: Molecular Virology

The Nidovirales represent a monophyletic but highly diverged order of plus-strand RNA viruses that currently comprises the families Coronaviridae, Mesoniviridae, Arteriviridae, Tobaniviridae and 10 other families of vertebrate and invertebrate viruses that share common genome organization and expression strategies. Viruses in this order include many important animal and human pathogens, with SARS-CoV, SARS-CoV-2, and MERS-CoV being prominent examples. With genome sizes of up to 41 kilobases (kb), nidoviruses feature the largest RNA virus genomes known to date. Nidoviruses have evolved an unusually complex set of enzymes involved in viral RNA synthesis that is unparalleled in the RNA virus world and believed to be required for the expansion of RNA genomes to unprecedented sizes and efficient replication in special ecological niches. The nidovirus replication/transcription complex (RTC) consists of a large number of virally encoded enzymes. Some of these enzymes are unique to specific nidovirus (sub)families and not conserved in other RNA viruses. This also includes a 3′-5′ exoribonuclease (ExoN) presumed to be involved in increasing the fidelity of nidovirus RNA synthesis. In the previous funding period, we characterized the biochemical properties of this enzyme for representative viruses of the Corona– and Tobaniviridae and extended our studies to other coronavirus nonstructural proteins (nsp7, 8, 9, 10, 12, 14) known to be essential components of the coronavirus RTC. Second, we identified and characterized two novel enzymatic activities that we confirmed to be essential for coronavirus replication, an RNA 3’-polyadenylyltranferase activity associated with nsp8 and a protein-specific NMP transferase activity associated with the NiRAN domain in nsp12. Third, we identified and characterized essential cis-active RNA elements involved in alphacoronavirus replication and, fourth, we embarked on the characterization of replicative proteins of other nidovirus families, focusing on the Mesoniviridae. In this context, we characterized the replicase polyprotein processing by the mesonivirus main protease, determined the crystal structure of this enzyme and identified the structural basis for its unusual substrate specificity. In the next funding period, we plan to advance our studies on the characterization of in vitro reconstituted nidovirus RTCs, involving corona- and mesonivirus protein complexes. We will investigate the mechanistic roles of protein-primed, RNA-primed and de novo initiation of viral RNA synthesis, focusing on the roles of specific viral proteins and cis-active RNA structural elements in these processes. Hypotheses derived from the in vitro studies will be validated by using genetically engineered human coronaviruses (HCoV-229E, SARS-CoV-2) and mesoniviruses as model systems. The studies will be based on biochemical approaches using recombinantly expressed proteins and protein complexes, RNA structure probing, and reverse-genetic approaches that are available in the laboratory or will be developed in the course of the project.

Project-related publications of the investigator:

  • Slanina H, Madhugiri R, Bylapudi G, Schultheiss K, Karl N, Gulyaeva A, Gorbalenya AE, Linne U, Ziebuhr J. 2021. Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit. Proc Natl Acad Sci U S A 118.
  • Shaban MS, Muller C, Mayr-Buro C, Weiser H, Meier-Soelch J, Albert BV, Weber A, Linne U, Hain T, Babayev I, Karl N, Hofmann N, Becker S, Herold S, Schmitz ML, Ziebuhr J, Kracht M. 2021. Multi-level inhibition of coronavirus replication by chemical ER stress. Nat Commun 12:5536.
  • Pfafenrot C, Schneider T, Muller C, Hung LH, Schreiner S, Ziebuhr J, Bindereif A. 2021. Inhibition of SARS-CoV-2 coronavirus proliferation by designer antisense-circRNAs. Nucleic Acids Res 49:12502-12516.
  • Krichel B, Bylapudi G, Schmidt C, Blanchet C, Schubert R, Brings L, Koehler M, Zenobi R, Svergun D, Lorenzen K, Madhugiri R, Ziebuhr J, Uetrecht C. 2021. Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes. Sci Adv 7.
  • Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, Haagmans BL, Lauber C, Leontovich AM, Neuman BW, Penzar D, Poon LLM, Samborskiy DV, Sidorov IA, Sola I, Ziebuhr J. 2020. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5:536-544.
  • Tvarogova J, Madhugiri R, Bylapudi G, Ferguson LJ, Karl N, Ziebuhr J. 2019. Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3′-Terminal Adenylyltransferase Activity. J Virol 93.
  • Kanitz M, Blanck S, Heine A, Gulyaeva AA, Gorbalenya AE, Ziebuhr J, Diederich WE. 2019. Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus. Virology 533:21-33.
  • Madhugiri R, Karl N, Petersen D, Lamkiewicz K, Fricke M, Wend U, Scheuer R, Marz M, Ziebuhr J. 2018. Structural and functional conservation of cis-acting RNA elements in coronavirus 5′-terminal genome regions. Virology 517:44-55.
  • Durzynska I, Sauerwald M, Karl N, Madhugiri R, Ziebuhr J. 2018. Characterization of a bafinivirus exoribonuclease activity. J Gen Virol 99:1253-1260.
  • Snijder EJ, Decroly E, Ziebuhr J. 2016. The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing. Adv Virus Res 96:59-126.
  • Madhugiri R, Fricke M, Marz M, Ziebuhr J. 2016. Coronavirus cis-Acting RNA Elements. Adv Virus Res 96:127-163.
  • Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C, Canard B, Ziebuhr J. 2006. Discovery of an RNA virus 3′->5′ exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc Natl Acad Sci U S A 103:5108-13.